The objective of this project is to define the initial, intracellular events of steroid hormone action. These events include steroid binding to the intracellular receptor molecule, "activation" of the receptor-steroid complex to a DNA-binding and nuclear-binding species, and binding of the activated complex to those nuclear acceptor sites involved in the regulation of transcription of specific genes. One approach that has been used to examine these steps is to compare the properties of agonist and antagonist receptor-steroid complexes. Using cell-free glucocorticoid receptors from a line of glucocorticoid responsive rat hepatoma tissue culture (HTC) cells that have been noncovalently bound by the reversible agonist dexamethasone or affinity labeled by the irreversible antiglucocorticoid dexamethasone 21-mesylate, we have found that there are minor, but real, physicochemical differences between the receptor complexes of glucocorticoid and antiglucocorticoid steroids. Thus, the rate of activation at O degrees C of the noncovalent agonist complexes was 2-fold faster than that of the covalent complexes and the strength of activated complex binding to calf thymus DNA was 2-fold higher for the noncovalent agonist complexes. This small difference in affinity for non-specific DNA was unchanged in the binding of activated noncovalent vs covalent complexes to the biologically active DNA sequences in the long termined repeat (LTR) of mouse mammary tumor virus (MMTV). Thus, the whole cell irreversible antiglucocorticoid activity of dexamethasone 21-mesylate cannot be ascribed to alterations in the affinity of the covalent complexes for biologically active or inactive DNA sequences or to changes in the rate of activation of the covalent complexes.